A specific binding assay is an assay that provides a biochemical test for detecting the presence or concentration of a substance in solutions that frequently contain a complex mixture of substances. An example of a specific binding assay is the immunoassay. Immunoassays rely on the ability of an antibody to bind with high specificity to one or a very limited group of molecules. A molecule that binds to an antibody is called an antigen. Immunoassays can be carried out to detect the presence in a solution of either member of an antigen/antibody pair. For antigen detection (i.e. where the antigen is the analyte), an antibody that specifically binds to that antigen can be prepared for use as an analytical reagent. When the analyte is a specific antibody its cognate antigen can be used as the analytical reagent. In either case the specificity of the assay depends on the degree to which the analytical reagent is able to bind to its specific binding partner to the exclusion of all other substances that might be present in the sample to be analyzed. In addition to the need for specificity, a binding partner must be selected that has a sufficiently high affinity for the analyte to permit an accurate measurement. The affinity requirements depend on the particular assay format that is used.
A competitive binding immunoassay is a particular example of the immunoassay. This may be better understood by considering the specific example of a test for a particular drug molecule, e.g. cocaine. In this case, a sample is obtained from a subject suspected of having taken the drug. This may be a saliva, blood or a urine sample. The sample is then mixed with a solution containing an antibody for that drug. Typically, these antibodies are labelled with some detectable marker, e.g. a gold particle, fluorescent marker, etc. If the drug is present, the labeled antibody molecules are all bound to the drug molecules. The mixture is then exposed to a test element to which drug molecules being tested for are bound. If the drug is present in the sample, then there will be no free labeled antibodies available to bind to the test element. No detectable change will occur. If, on the other hand, no drug is present in the sample, free labeled antibodies will be available and will bind to the test element. A detectable change will occur. The skilled person will appreciate that, rather than binding the drug molecules themselves to the test element, analogues of the drug molecule may be bound, i.e. molecules possessing the relevant binding group or groups.
Competitive binding immunoassays may be performed using a lateral flow device. Such a test methodology is described in GB Patent 2339615 of Cozart Bioscience Limited, corresponding published International Application WO 00/04381, and Journal of Forensic Science 2001, volume 46, pages 1214-1220. A common feature of lateral flow devices for analyte detection is the provision of a test strip or sheet comprising a dry porous material such as nitrocellulose through which a liquid sample can be drawn to reach one or more spatially distinct analyte detection zones. Each such zone presents an immobilised specific binding reagent.
FIG. 1 illustrates schematically a test strip for use in carrying out a lateral flow immunoassay in which reference numeral 1 identifies a porous strip of nitrocellulose sheet laminated onto a backing support, numeral 2 identifies the analyte detection zone presenting immobilised analyte (or an analogue of the analyte), numeral 3 identifies a control zone presenting immobilised antibody to capture labelled antibody, numeral 4 is a label release pad which releases labelled antibody into liquid drawn into this pad from the sample receiving pad, numeral 5, and numeral 6 identifies a wicking pad.
GB2339615 also describes apparatus for reading results of tests carried out using a lateral flow test strip of the type illustrated in FIG. 1. According to the described approach, test strips are integrated into a disposable cartridge, the disposable cartridge holder comprising a swab holder for receiving the end of a swab onto which a test sample (e.g. saliva) has been placed. To perform a test, the swab holder is filled with a buffer solution and the swab inserted. (The swab holder may be pre-filled and the top sealed with a removable foil lid.) The test sample mixes with the buffer solution and is soaked onto and through the test strip. The cartridge is inserted into a reader and, following some incubation period, e.g. 5 minutes, an optical reading performed.
Results are displayed on a digital display. Prior art immunoassays of the type described in GB2339615 rely upon a pre-mixing of the test sample with a buffer solution, and a subsequent washing of the solution through the zone containing the labelled antibody and into the test strip.
US patent application publication number US2006/0275922 describes a lateral flow immunoassay test device that facilitates a first pre-mixing of a sample with a buffer, and a second pre-mixing of the mixed sample and buffer with a dry reagent which may be an antigen or a labelled antibody.
An alternative to the competitive binding immunoassay is the so-called sandwich binding immunoassay. In such sandwich assays, binding of the analyte occurs at a minimum of two sites by two antibody moieties. The first moiety is labelled as a reagent whilst the second is immobilised to the test strip. Therefore, the appearance of a visual indication is an indication of a positive test result.